Use of prion conversion modulating agents

ABSTRACT

The use of Apolipoprotein B, Apolipoprotein E, fragments and mimetics thereof is provided for diagnostic, detection, prognostic and therapeutic applications In prion diseases. More specifically, the invention provides the use of Apolipoprotein B or fragments thereof for modulating or identifying modulators of the prion protein replication which are implicated in the pathogenesis of transmissible spongiform encephalopathics and other prion diseases.

FIELD OF THE INVENTION

This invention relates to the use of apolipoprotein B or apolipoproteinE or fragments or mimetics thereof for diagnostic, detection, prognosticand identifying modulators of the prion protein replication. Morespecifically, the invention provides the use of modulators ofapolipoprotein B or fragments thereof for modulating the prion proteinreplication which are implicated in the pathogenesis of transmissiblespongiform encephalopathies and other prion diseases.

BACKGROUND OF THE INVENTION

Creutzfeldt-Jakob disease (CJD) in humans and scrapie and bovinespongiform encephalopathy (BSE) in animals are some of the diseases thatbelong to the group of Transmissible Spongiform Encephalopathies (TSE),also known as prion diseases (Prusiner, 1991). These diseases arecharacterized by an extremely long incubation period, followed by abrief and invariably fatal clinical disease (Roos et al., 1973). To dateno therapy is available.

Although these diseases are relatively rare in humans, the risk for thetransmissibility of BSE to humans through the food chain has seized theattention of the public health authorities and the scientific community(Soto at al., 2001). Variant CJD (vCJD) is a new disease, which wasfirst described in March 1996 (Will et al., 1996). In contrast totypical cases of sporadic CJD (sCJD), this variant form affects youngpatients (average age 27 years old) and has a relatively long durationof illness (median 14 months vs. 4.5 months in traditional CJD). A linkbetween vCJD and BSE was first hypothesized because of the associationof these two TSEs in place and time (Bruce, 2000). The most recent andpowerful evidence comes from studies showing that the transmissioncharacteristics of BSE and vCJD to mice are almost identical andstrongly indicating that they are due to the same causative agent (Bruceet al., 1997). Moreover, transgenic mice carrying a human or a bovinegene have now been shown to be susceptible to BSE and vCJD (Scott etal., 1999). Furthermore, no other plausible hypothesis for theoccurrence of vCJD has been proposed and intensive CJD surveillance infive European countries, with a low exposure to the BSE agent, hasfailed to identify any additional cases. In conclusion, the most likelycause of vCJD is exposure to the BSE agent, probably due to dietarycontamination with affected bovine central nervous system tissue.

The nature of the transmissible agent has been matter of passionatecontroversy. Further research, has indicated that the TSE agent differssignificantly from viruses and other conventional agents in that itseems not to contain nucleic acids (Prusiner, 1998). Additionally, thephysicochemical procedures that inactivate most viruses, such asdisrupting nucleic acids, have proved ineffective in decreasing theinfectivity of the TSE pathogen. In contrast, the procedures thatdegrade protein have been found to inactivate the pathogen (Prusiner,1991). Accordingly, the theory that proposes that the transmissibleagent is neither a virus nor other previously known infectious agent,but rather an unconventional agent consisting only of a protein recentlygained widespread acceptability (Prusiner, 1998). This new class ofpathogen was called a “prion”, short for “proteinaceous infectiousparticle”. In TSE, prions are composed mainly of a misfolded proteinnamed PrP^(Sc)(for scrapie PrP), which is a post-translationallymodified version of a normal protein, termed PrP^(C) (Cohen et al.,1998). Chemical differences have not been detected to distinguish thesetwo PrP isoforms and the conversion seems to involve a conformationalchange whereby the α-helical content of the normal protein diminishesand the amount of β-sheet increases (Pan et al., 1993). The structuralchanges are followed by alterations in the biochemical properties:PrP^(C) is soluble in non-denaturing detergents, PrP^(Sc) is insoluble;PrP^(C) is readily digested by proteases (also called protease sensitiveprion protein) while PrP^(Sc) is partially resistant, resulting in theformation of a N-terminally truncated fragment known as PrP^(C) is(protease resistant prion protein) (Cohen et al., 1998).

The notion that endogenous PrP^(C) is involved in the development ofinfection is supported by experiments in which endogenous PrP gene wasknocked out where the animals were both resistant to prion disease andunable to generate new infectious particles (Bueler et al., 1993). Inaddition, it is clear that during the tune between the inoculation withthe infectious protein and the appearance of the clinical symptoms,there is a dramatic increase in the amount of PrP^(Sc).

These findings suggest that endogenous PrP^(C) is converted to PrP^(Sc)conformation by the action of an infectious form of the PrP molecule(Soto et al., 2001). Prion replication is hypothesized to occur whenPrP^(Sc) in the infecting inoculum interacts specifically with hostPrP^(C), catalyzing its conversion to the pathogenic form of theprotein. A physical association between the two isoforms during theinfectious process is suggested by the primary sequence specificity inprion transmission (Telling et al., 1994) and by the reported in vitrogeneration of PrP^(Sc)-like molecules by mixing purified PrP^(C) withPrP^(Sc) (Saborio et al., 2001). However, the exact mechanism underlyingthe conversion is not known.

Investigations with chimeric transgenes showed that PrP^(Sc) andPrP^(Sc) are likely to interact within a central domain delimited bycodons 96 and 169 (Prusiner, 1996) and synthetic PrP peptides spanningthe region 109-141 proved to be able to bind to PrP^(C) and compete withPrP^(Sc) interaction (Chabry et al., 1998).

Based on data with transgenic animals, it has been proposed thatadditional brain factors present in the host are essential for prionpropagation (Telling et al., 1995). It has been demonstrated previouslythat prion conversion does not occur under experimental conditions wherepurified PrP^(C) and PrP^(Sc) are mixed and incubated (Saborio et al.,1999) but that the conversion activity is recovered when the bulk ofcellular proteins are added back to the sample (Saborio et al., 1999).This finding provides direct evidence that other factors present in thebrain are essential to catalyse prion propagation.

The observation that cholesterol depletion decreases the formation ofPrP^(Sc) whereas sphingolipid depletion increases PrP^(Sc) formation,suggested that “lipid rafts”(lipid domains in membranes that containsphingolipids and cholesterol) may be the site of the PrP^(c) toPrP^(Sc) conversion reaction involving either a raft-associated proteinor selected raft lipids (Fantini et al., 2002). However, the role oflipid rafts in prion infectivity is still unclear.

Several in vitro methods of detections of prions in a sample have beendeveloped. The set of known detection methods, include PrP^(Sc)detection methods using specific ligand carriers selected fromaminoglycans, fibronectin and Apolipoprotein A (WO 02/065133); methodsusing the monoclonal antibodies selected from Gö138, 3B5 and 12F10(Schulz et al., 2000); methods based on the formation of a complexbetween PrP^(Sc) and Apolipoprotein H (WO 03/005037); or methods basedon the PrP^(Sc) in vitro amplification called protein misfolding cyclicamplification (PMCA) described in Saborio et al., 2001 and Lucassen etal., 2003.

Apolipoprotoin B is the major protein component of the two knownatherogenic lipoproteins, Low Density Lipoproteins (LDL) and remnants oftriglyceride-rich lipoproteins. The apolipropotein B concentration isconsidered to be a direct reflection of the number of atherogenicparticles in the blood and has been proposed as a parameter fordetermining the risk of atherosclerosis.

Apolipoprotein E is a constituent of several plasma lipoprotein such aschylomicrons, very low-density lipoproteins (VLDL), and high-densitylipoproteins (HDL) (Lehninger et al., 1993).

Apolipoprotein E has recently emerged as a major genetic risk factor forAlzheimer's disease, a neurodegenerative disorder (U.S. Pat. No.6,022,683) and upregulated in the cerebrospinal fluid of patients withvariant CJD and Alzheimer's disease compared to patients with sporadicCJD (Choe et al., 2002). The Apolipoprotein E 4/4 phenotype isassociated with increased risk of coronary heart diseases andCreutzfeld-Jakob disease (Golaz et al., 1995). Apolipoprotein E geneexpression was found to be increased in astrocytes associated with theneuropathological lesions in a scrapie animal model (Dietrich et al.,1991).

Apolipoprotein E was found to recognise a shared structural motif ofamyloids and prion which, after induction, can accelerate the adoptionof a beta-sheet conformation (Baumann et al., 2000).

Apolipoprotein B and E are ligands for the LDL receptor and are knownfor its prominent role in cholesterol transport and plasma lipoproteinmetabolism via LDL receptor interactions (Segrest et al, 2001; Clavey etal, 1991).

One approach to the treatment and prevention of prion diseases has beento develop agents for blocking the transformation of PrP^(c) intoPrP^(Sc). Some agents proposed were Congo red dye (U.S. Pat. No.5,276,059), nerve growth peptides (U.S. Pat. No. 5,134,121), fragmentsof prion proteins (U.S. Pat. No. 6,355,610), compounds that reducesApolipoprotein E release in the brain tissue (US 2002/0155426),therapeutic agents that prevent Apolipoprotein E4 to interact withneuronal LDL receptor-related protein (WO 97/14437), compounds thatincrease Apolipoprotein E levels (WO 99/15159) and beta-sheet breakerpeptides (U.S. Pat. No. 5,948,763).

It would be desirable to develop new methods for identifying andinhibiting the prion conversion factor(s).

SUMMARY OF THE INVENTION

It is an object of the invention to provide a use of peptides orproteins in an assay for the detection of PrP^(Sc) formation in asample.

It is also an object of the invention to provide a use of peptides orproteins in a screening assay for identifying compounds that modulatethe conversion of PrP^(c) into PrP^(Sc).

It is further an object of the invention to provide a substance which issuitable for the treatment of, and/or prevention of, and/or delaying theprogression of prion related disorders, notably, bovine spongiformencephalopathy (BSE) and Creutzfeld-Jacob Disease (CJD).

In a first aspect, the invention provides a use of a peptide or aprotein selected from Apolipoprotein B; a fragment or mimetic thereof,Apolipoprotein E and a fragment or mimetic thereof, in an assay for thedetection of PrP^(Sc) formation in a sample.

In a second aspect, the invention provides a use of a peptide or aprotein selected from Apolipoprotein B; a fragment or mimetic thereof,Apolipoprotein E and a fragment or mimetic thereof, in a screening assayfor identifying compounds that modulate the conversion of PrP^(c) intoPrP^(Sc).

In a third aspect, the invention provides a use of a modulator,preferably an inhibitor or an antagonist, of a peptide or a protein,wherein the peptide or the protein is selected from Apolipoprotein B; afragment and a mimetic thereof, for the preparation of a pharmaceuticalcomposition for the treatment of a prion disease, notably, bovinespongiform encephalopathy (BSE) and a Creutzfeld-Jacob Disease (CJD).

In a fourth aspect, the invention provides a method for the diagnosis ordetection of a prion disease within a subject suspected of sufferingfrom such a disease which comprises (i) contacting a sample from saidsubject with a peptide or a protein selected from Apolipoprotein B; afragment or a mimetic thereof. Apolipoprotein E; a fragment thereof anda mimetic thereof; (ii) contacting the sample obtained from step (i)with PrP^(C) or PrP^(C) containing mixtures, such as brain homogenates,cell lysates, lipid rafts preparation; and (iii) determining thepresence and/or amount of PrP^(Sc) in said sample.

In a fifth aspect, the invention provides a method of determining amarker that predisposes a subject to a prion disease, comprising (i)measuring a level of a protein selected from Apolipoprotein B and afragment thereof, and (ii) correlating said level of protein obtained insaid measuring step with the occurrence of a prion disease.

In a sixth aspect, the invention provides a method for the detection ofPrP^(Sc) formation within a sample, which assay comprises (i) contactingsaid sample with a peptide or a protein selected from Apolipoprotein B;a fragment or a mimetic thereof; Apolipoprotein E; a fragment thereofand a mimetic thereof (ii) contacting the sample obtained from step (i)with PrP^(C) or PrP^(C) containing mixtures, such as brain homogenates,cell lysates, lipid rafts preparation; and (iii) determining thepresence and/or amount of PrP^(Sc) in said sample.

In a seventh aspect, the invention provides a method for identifying acompound which modulates, preferably inhibits or antagonizes, thetransition of PrP^(C) into PrP^(Sc) comprising: (i) contacting saidsample with a peptide or a protein selected from Apolipoprotein B; afragment or a mimetic thereof, Apolipoprotein E; a fragment thereof anda mimetic thereof (a) in the presence of said compound and (b) in theabsence of said compound; (ii) contacting the sample obtained from step(i) a and (i) b with PrP^(C) or PrP^(C) containing mixtures, such asbrain homogenates, cell lysates, lipid rafts preparation; and (iii)determining the amount of PrP^(Sc) (a) in the presence of said compoundand (b) in the absence of said compound.

In a eighth aspect, the invention provides an assay for the detection ofPrP^(Sc) formation within a sample, which assay comprises (i) contactingsaid sample with a peptide or a protein selected from Apolipoprotein B;a fragment or a mimetic thereof; Apolipoprotein E; a fragment thereofand a mimetic thereof (ii) contacting the sample obtained from step (i)with PrP^(C) or PrP^(C) containing mixtures, such as brain homogenates,cell lysates, lipid rafts preparation; and (iii) determining thepresence and/or amount of PrP^(Sc) in said sample.

In a ninth aspect, the invention provides a screening assay foridentifying a compound which modulates, preferably inhibits orantagonizes, the transition of PrP^(C) into PrP^(Sc) comprising: (i)contacting said sample with a peptide or a protein selected fromApolipoprotein B; a fragment or a mimetic thereof; Apolipoprotein E; afragment thereof and a mimetic thereof (a) in the presence of saidcompound and (b) in the absence of said compound; (ii) contacting thesample obtained from step (i) a and (i) b with PrP^(C) or PrP^(C)containing mixtures, such as brain homogenates, cell lysates, lipidrafts preparation; and (iii) determining the amount of PrP^(Sc) (a) inthe presence of said compound and (b) in the absence of said compound.

DETAILED DESCRIPTION OF THE INVENTION

The following paragraphs provide definitions of various terms, and areintended to apply uniformly throughout the specification and claimsunless an otherwise expressly set out definition provides a differentdefinition.

The term “Gerstmann-Strassler-Scheinker Disease” abbreviated as “GSS”refers to a form of inherited human prion disease. The disease occursfrom an autosomal dominant disorder. Family members who inherit themutant gene succumb to GSS.

The term “prion” shall mean a transmissible particle known to cause agroup of such transmissible conformational diseases (spongiformencephalopathies) in humans and animals. The term “prion” is acontraction of the words “protein” and “infection” and the particles arecomprised largely if not exclusively of PrP^(Sc) molecules.

“Prions” are distinct from bacteria, viruses and viroids. Known prionsinclude those which infect animals to cause scrapie, a transmissible,degenerative disease of the nervous system of sheep and goats as well asbovine spongiform encephalopathies (USE) or mad cow disease and felinespongiform encephalopathies of cats. Four prion diseases known to affecthumans are Kuru, Creutzfeldt-Jakob Disease (CJD),Gerstmann-Strassler-Scheinker Disease (GSS), and fatal filial insomnia(FFI) (Prusiner, 1991). As used herein prion includes all forms ofprions causing all or any of these diseases or others in any animalsused—and in particular in humans and in domestic farm animals.

The term “lipid rafts” refers to small platforms, composed ofsphingolipids and cholesterol in the outer exoplasmic layer, connectedto Cholesterol in the inner cytoplasmic layer of the bilayer that havebeen reviewed recently (Simons et al., 2000). Lipid rafts can beisolated as they are insoluble in certain detergents such as tritonX-100 at 4° C. Therefore, rafts can be purified as detergent-insolublemembranes (DIMs) or detergent-resistant membranes (DRMs) byultracentrifugation on sucrose gradients. Rafts are enriched inGPI-anchored proteins, as well as proteins involved in signaltransduction and intracellular trafficking. In neurons, lipid rafts actas platforms for the signal transduction initiated by several classes ofneurotrophic factors (Tsui-Pierchala et al., 2002). Example for lipidrafts extraction is given in Example n°2 §c.

The term “prion conversion factor”refers to a factor comprisingproteins, lipids, enzymes or receptors that acts as a co-factor orauxiliary factor involved in the process of conversion of PrP^(C) intoPrP^(Sc) and favors the onset and/or progression of the prion disease.

The terms “standardized prion preparation”, “prion preparation” and thelike are used interchangeably herein to describe a compositioncontaining prions which composition is obtained for example from braintissue of mammals substantially the same genetic material as relates toPIP proteins, e.g. brain tissue from a set of mammals which exhibitsigns or prion disease or for example a composition which is obtainedfrom chronically prion infected cells.

The terms “sensitive to infection”, “sensitive to prion infection” andthe like are use for a material from a mammal, including cells, that canbe infected with an amount and type of prion which would be expected tocause prion disease or symptoms.

By analogy, the terms “resistant to infection”, “resistant to prioninfection” and the like are used for a material from a mammal, includingcells which has the characteristic to be resistant when infected with anamount and type of prion which would be expected to cause prion diseaseor symptoms and remain uninfected even after several infective prionmaterial inoculations.

The term “sample” refers to a biological extract from a mammal,including cell sample, body fluid, genetic material such as brainhomogenate, cells, lipid rafts or purified peptides and proteins.

The term “incubation time” shall mean the time from inoculation of ananimal with a prion until the time when the animal first developsdetectable symptoms of disease resulting from infection, it also meansthe time from inoculation of material from a mammal e.g. brainhomogenate, cells, lipid rafts from cells, with prion until the timewhen the prion infection is detectable such as through the conversion ofPrP^(C) into PrP^(Sc). Several methods of detection of prion infectionand PrP conversion are known by a person skilled in the art.

The terms “fraction” or “fragment” refer to any fragment of thepolypeptidic chain of the compound itself, alone or in combination withrelated molecules or residues bound to it, for example residues ofsugars or phosphates, or aggregates of the original polypeptide orpeptide. Such molecules can result also from other modifications whichdo not normally alter primary sequence, for example in vivo or in vitrochemical derivativization of peptides (acetylation or carboxylation),those made by modifying the pattern of phosphorylation (introduction ofphosphotyrosine, phosphoserine, or phosphothreonine residues) orglycosylation (by exposing the peptide to enzymes which affectglycosylation e.g., mammalian glycosylating or deglycosylating enzymes)of a peptide during its synthesis and processing or in furtherprocessing steps.

The terms “modulator” or “modulatory compound” refer to molecules thatmodify the functions and/or properties (such as receptor binding, lipidaffinity, enzyme interaction, structural arrangement, synthesis,metabolism) of the natural protein “Modulators” or “modulatorycompounds”include “agonists” and antagonists”. Modulators” includepeptides, proteins or fragments thereof, peptidomimetics, organiccompounds and antibodies.

The term “mimetic” refer to molecules that mimic the functions and/orproperties (such as receptor binding, lipid affinity, enzymeinteraction, structural arrangement, synthesis, metabolism) of a naturalprotein. These compounds have for example the property to either enhancea property of the natural protein (i.e. to lead to the same activitywhen the compound is added to the natural protein as obtained with anincrease in concentration in the natural protein) or to exhibit the sameproperty as a natural protein (i.e. to lead to the same activity whenthe compound replaces the natural protein). “Mimetics” include peptides,proteins or fragments thereof, peptidomimetics and organic compounds.Examples of Apolipoprotein E mimetics are described in US 20020128175and WO 2004043403.

The terms “inhibitor” or “antagonist” refer to molecules that alterpartially or impair the functions and/or properties (such as receptorbinding, lipid affinity, enzyme interaction, structural arrangement,synthesis, secretion, metabolism) of the natural protein. “Inhibitors”or “antagonists” include peptides, proteins or fragments thereof,peptidomimetics, organic compounds and antibodies. Examples ofApolipoprotein B antibodies are described in Choi et al., 1997 and inWang et al., 2000. Examples of Apolipoprotein antagonists can beantagonists that alter or impair the role of Apolipoproteins B or E inthe cholesterol transport pathway. Examples of compounds that alterApolipoprotein B secretion or synthesis are described in U.S. Pat. No.6,369,075, U.S. Pat. No. 6,197,972, WO 03/002533 and WO 03/045921. Other“modulators” or “antagonists” can be modulators of the LDL receptor,preferably LDL-receptor antagonists such as anti-LDL receptorantibodies. Examples of monoclonal antibodies to the LDL receptor aregiven in WO 01/68710.

The term “protein misfolding cyclic amplification assay” or “YMCA assay”is an assay that for the diagnosis or detection of conformationaldiseases which comprises a cyclic amplification system to increase thelevels of the pathogenic conformer such as described for example in WO02/04954.

The term “maker” for a disease refers to a biological parameter or valueincluding a genetic character, inherited protein mutation(s), bloodlevel of a protein or an enzyme that is different from the average valuein a heterogeneous population of individuals and whose occurrencecorrelates with the occurrence of said disease with a statisticalsignificance. A “marker” for a disease or condition is typically definedas a certain cut, off level of a said biological variable. A “marker”provides basis for determining the risk (probability of occurrence) of adisease in a subject.

The term “complex” includes the formation of an entity by theinteraction of several molecules, several proteins, several peptidestogether or with a receptor. These interactions may be reversible and/ortransient. These interactions may induce changes in the properties ofthe interacting molecules, proteins, peptides or receptors.

By “effective amount”, it is meant a concentration of peptide(s) that iscapable of slowing down or inhibiting the formation of PrP^(Sc)deposits, or of dissolving preformed deposits. Such concentrations canbe routinely determined by those of skill in the art. It will also beappreciated by those of skill in the art that the dosage may bedependent on the stability of the administered peptide. A less stablepeptide may require administration in multiple doses.

The preparation of antibodies is known by the person skilled in the art.It is referred by “antibody” to a monoclonal antibody, chimericantibody, humanized antibody, anti-anti-Id antibody or fragment thereofwhich specifically recognises and binds to Apo B or Apo E and fragmentsthereof. For example, monoclonal antibodies are obtained though thegeneration of hybridoma cells lines producing monoclonal antibodiescapable of specifically recognising and binding Apo B and/or fragmentsthereof. More specifically, these monoclonal antibodies are capable ofspecifically recognising and binding Apo B. A monoclonal antibody can beprepared in a conventional manner, e.g. by growing a cloned hybridomacomprising a spleen cell from a mammal immunized with hApo B and ahomogenic or heterogenic lymphoid cell in liquid medium or mammalianabdomen to allow the hybridoma to produce and accumulate the monoclonalantibody. Preferably, the antibody specifically recognises and binds toApo B-LDL recognizing fragments.

The present invention provides compounds capable of controlling,including increasing and/or inhibiting, the conversion of PrP^(C) intoPrP^(Sc) in prion diseases.

The activity of the compounds of the invention in controlling theconversion of PrP^(C) into PrP^(Sc) in prion diseases can be detectedusing, for example, an in vitro assay, such as that described by Saborioet al., 2001 which measures the ability of compounds of the invention tomodulate the conversion of PrP^(C) into PrP^(Sc). Results are reportedin the Examples.

In one embodiment, the invention provides a use of a peptide or aprotein selected from Apolipoprotein B; a fragment thereof or a mimeticthereof; Apolipoprotein B; a fragment thereof and a mimetic thereof,preferably Apolipoprotein B; a fragment thereof and a mimetic thereof;in an assay for the detection of pipe formation in a sample.

In one further embodiment of the invention, the peptide or the proteinselected from Apolipoprotein B; a fragment thereof or a mimetic thereof,Apolipoprotein E; a fragment thereof and a mimetic thereof, preferablyApolipoprotein B or a fragment thereof; used in an assay for thedetection of PrP^(Sc) formation in a sample binds and/or forms a complexwith the LDL receptor.

In another embodiment, the invention provides a use of a peptide or aprotein selected from Apolipoprotein B; a fragment thereof or a mimeticthereof; Apolipoprotein E; a fragment thereof and a mimetic thereof,preferably Apolipoprotein B or a fragment thereof, in a screening assayfor the identifying compounds that modulate the conversion of PrP^(c)into PrP^(Sc).

In another further embodiment of the invention, the peptide or theprotein selected from Apolipoprotein B; a fragment thereof or a mimetic;Apolipoprotein E; a fragment thereof and a mimetic thereof, preferablyApolipoprotein B or a fragment thereof, thereof, is used in a screeningassay for the identifying compounds that modulate the conversion ofPrP^(c) into PrP^(Sc) binds and/or forms a complex with the LDLreceptor.

In a further embodiment of the invention, the assay is a ProteinMisfolding Cyclic (PMCA) assay.

In a preferred embodiment of the invention, the Protein MisfoldingCyclic (PMCA) assay uses normal brain homogenate as a source of normalPrP^(c) and prion conversion factor.

In a further embodiment of the invention, the protein according to theinvention is Apolipoprotein B.

In a preferred embodiment of the invention, the Protein MisfoldingCyclic (PMCA) assay uses cell lysates or lipid rafts extracted fromprion infection sensitive neuroblasma cells, such as line N2a, describedin Example 2, and equivalent, as a source of normal PrP^(c) and prionconversion actor. Lipid raft fractions can also be purified directlyfrom the brain to serve as a source of substrate for PMCA.

In a preferred embodiment, the invention provides a use ofApolipoprotein Bin an assays for the detection of PrP^(C) in a sample,wherein the assay is a Protein Misfolding Cyclic Amplification (PMCA)assay using lipid rafts from infection sensitive neuroblasma cell lineN2a as a source of normal PrP^(C) and substrate.

In another embodiment, the invention provides a use of a modulator,preferably an inhibitor or an antagonist, of a peptide or a protein,wherein the peptide or the protein is selected from Apolipoprotein B; afragment thereof or a mimetic thereof for the preparation of apharmaceutical composition for the treatment of a prion disease,notably, bovine spongiform encephalopathy (BSE) and Creutzfeld-JacobDisease (CJD). The modulator modifies for example the functions and/orproperties of Apolipoprotein B or of a fragment thereof.

In a further embodiment of the invention, the modulator, preferably aninhibitor or an antagonist, of a peptide or a protein, wherein thepeptide or the protein is selected from Apolipoprotein B; a fragmentthereof and a mimetic thereof which modifies, preferably inhibits thebinding and/or the formation of a complex between Apolipoprotein B andthe LDL receptor. An example of such modulator can be a LDL receptormodulator, such as a LDL-receptor antagonist such as an anti-LDLreceptor antibody.

In a preferred embodiment of the invention, the modulator is anantagonist to Apolipoprotein B or a fragment thereof.

In a further preferred embodiment of the invention, the modulator is anantibody raised against Apolipoprotein B or against a fragment thereof.

In another preferred embodiment of the invention, the modulator is anantibody raised against Apolipoprotein B.

In another preferred embodiment of the invention, the modulator is anantibody raised against a fragment of Apolipoprotein B, which fragmentis of, or about, a molecular weight selected from 30, 35 and 40 kDa.

In another preferred embodiment of the invention, the modulator is anantibody raised against a fragment of Apolipoprotein B, which fragmentcomprises a sequence selected from fragments taken between positions3201-3558, 3548-3905, 3201-3905, 3291-3558, 3548-3815 and 3291-3815.

In a preferred embodiment of the invention, the peptide or protein isselected from Apolipoprotein B or a fragment thereof.

In a preferred embodiment of the invention, the peptide or proteincontains the sequence of SEQ D NO: 3.

In another preferred embodiment of the invention, the peptide or proteinis a fragment which is of, or about, a molecular weight selected from30, 35 and 40 kDa.

In another preferred embodiment of the invention, the peptide or proteinis a fragment of Apolipoprotein B, comprising a sequence selected fromfragments, taken between positions 3201-3558, 3548-3905, 3201-3905,3291-3558, 3548-3815 and 3291-3815.

In an embodiment of the invention, the invention provides a method forthe diagnosis or detection of a prion disease within a subject suspectedof suffering from such a disease which comprises (i) contacting a samplefrom said subject with a peptide or a protein selected fromApolipoprotein B; a fragment or a mimetic thereof, Apolipoprotein E; afragment thereof and a mimetic thereof preferably Apolipoprotein B or afragment thereof, (ii) contacting the sample obtained from step (i) withPrP^(C) or PrP^(C) containing mixtures, such as brain homogenates, celllysates, lipid rafts preparation; and (ii) determining the presenceand/or amount of PrP^(Sc) in said sample. The sample from the subjectincludes a biological extract from a mammal such as cell sample, geneticmaterial, body fluid, brain homogenate, cells and lipid rafts.

In another embodiment of the invention, the invention provides a methodof determining a marker that predisposes a subject to a prion disease,comprising (i) measuring a level of a protein selected fromApolipoprotein B and a fragment thereof in said sample; (ii) contactingthe sample obtained from step (i) with PrP^(C) or PrP^(C) containingmixtures, such as brain homogenates, cell lysates, lipid raftspreparation; and (iii) correlating said level of protein obtained insaid measuring step with the occurrence of a prion disease. The makerincludes a biological parameter or value such as a genetic character,inherited protein mutation(s), blood level of a protein or an enzyme.

In another embodiment of the invention, the invention provides a methodfor the detection of PrP^(Sc) formation within a sample, which assaycomprises (i) contacting said sample with a peptide or a proteinselected from Apolipoprotein B; a fragment thereof or a mimetic thereof,Apolipoprotein E, a fragment thereof and a mimetic thereof, preferablyApolipoprotein B or a fragment thereof (ii) contacting the sampleobtained from step (i) with PrP^(C) or PrP^(C) containing mixtures, suchas brain homogenates, cell lysates, lipid rafts preparation; and (iii)determining the presence and/or amount of PrP^(Sc) in said sample. Thesample can be a biological preparation for which the presence of prionis to be detected for quality control reasons and/or a sample extractedfrom a subject that is suspected of suffering of such a disease,including a biological extract from a mammal such as cell sample,genetic material, body fluid, brain homogenate, cells and lipid rafts.

In another embodiment of the invention, the invention provides a methodfor identifying, in a sample, a compound which modulates, preferablyinhibits or antagonizes, the transition of PrP^(C) into PrP^(Sc)comprising: (i) contacting said sample with a peptide or a proteinselected from Apolipoprotein B; a fragment thereof or a mimetic thereof,Apolipoprotein E, a fragment thereof and a mimetic thereof, preferablyApolipoprotein B or a fragment thereof (ii) contacting the sampleobtained from step (i) (a) in the presence of said compound and (b) inthe absence of said compound; (iii) contacting the sample obtained fromstep (i) a and (i) b, with PrP^(C) or PrP^(C) containing mixtures, suchas brain homogenates, cell lysates, lipid rafts preparation; and (iv)determining the amount of PrP^(Sc) (a) in the presence of said compoundand (b) in the absence of said compound. The modulator, includesantibodies, inhibitors of Apolipoproteins B binding, including bindingto the LDL receptor, and/or secretion and/or synthesis.

Still another embodiment of the present invention, is a method fortreating or preventing a prion disease such as bovine spongiformencephalopathy (BSE) and Creutzfeld-Jacob Disease (CJD), wherein themethod comprises administering an effective dose of the above-mentionedmodulator of a peptide or a protein, wherein the peptide or the proteinis selected from Apolipoprotein B and a fragment thereof, to a subjectin the need thereof, wherein the subject can be human or animal.

In a preferred method of use of the modulators, preferably inhibitors,administration of the modulators is by injection or infusion, atperiodic intervals. The administration of a compound of the inventionmay begin before any symptoms are detected in the patient, and shouldcontinue thereafter.

The above-mentioned modulatory compounds of the present invention may beadministered by any means that achieves the intended purpose. Forexample, administration may be by a number of different routesincluding, but not limited to subcutaneous, intravenous, intradermal,intramuscular, intraperitoneal, intra-cerebral, intrathecal, intranasal,oral, rectal, transdermal, intranasal or buccal. Preferably thecompounds of the invention are administered by subcutaneous,intramuscular or intravenous injection or infusion.

Parenteral administration can be by bolus injection or by gradualperfusion over time. A typical regimen for preventing, suppressing, ortreating prion related disorders, comprises either (1) administration ofan effective amount in one or two doses of a high concentration ofmodulatory in the range of 0.5 to 10 mg of peptide, more preferably 0.5to 10 mg of peptide, or (2) administration of an effective amount of thepeptide in multiple doses of lower concentrations of modulatorycompounds in the range of 10-1000 μg, more preferably 50-500 μg over aperiod of time up to and including several months to several years. Itis understood that the dosage administered will be dependent upon theage, sex, health, and weight of the recipient, concurrent treatment, ifany, frequency of treatment, and the nature of the effect desired. Thetotal dose required for each treatment may be administered by multipledoses or in a single dose.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions, which may containauxiliary agents or excipients which are known in the art. Suitableformulations for parenteral administration include aqueous solutions ofthe active compounds in water-soluble form, for example, water-solublesalts. In addition, suspension of the active compound as appropriateoily injections suspensions may be administered.

In another embodiment of the invention is provided an assay for thedetection of the formation of PrP^(Sc) within a sample, which assaycomprises (i) contacting said sample with a peptide or a proteinselected from Apolipoprotein B; a fragment thereof or a mimetic thereof,Apolipoprotein E, a fragment thereof and a mimetic thereof, preferablyApolipoprotein B or a fragment thereof (iii) contacting the sampleobtained from step (iii) contacting the sample obtained from step (ii)with PrP^(C) or PrP^(C) containing mixtures, such as brain homogenates,cell lysates, lipid rafts preparation; and (iv) determining the presenceand/or amount of PrP^(Sc) in said sample. The sample can be a biologicalpreparation for which the presence of prion is to be detected forquality control reasons and/or a sample extracted from a subject that issuspected of suffering of such a disease, including a biological extractfrom a mammal such as cell sample, genetic material, body fluid,including blood, serum, plasma, brain homogenate, cells and lipid rafts.

In another embodiment of the invention, is provided a screening assayfor identifying a compound which modulates, preferably inhibits orantagonizes, the transition of PrP^(C) into PrP^(Sc) comprising: (i)contacting said sample with a peptide or a protein selected fromApolipoprotein B; a fragment thereof or a mimetic thereof;Apolipoprotein E; a fragment thereof or a mimetic thereof, preferablyApolipoprotein B or a fragment thereof (a) in the presence of saidcompound and (b) in the absence of said modulatory compound, (ii)contacting the sample obtained from step (i) a and (i) b with PrP^(C) orPrP^(C) containing mixtures, such as brain homogenates, cell lysates,lipid rafts preparation; and (iii) determining the amount of PrP^(Sc)(a) in the presence of said compound and (b) in the absence of saidmodulatory compound. The modulator, includes antibodies, inhibitors ofApolipoproteins B and/or secretion and/or synthesis.

In further embodiment of the invention, is provided a diagnostic kit foruse in the assay of the invention, comprising a probe for receiving asample and a peptide or a protein selected from Apolipoprotein B; afragment thereof and a mimetic thereof; Apolipoprotein E, a fragmentthereof and a mimetic thereof. The kit of the invention comprises kitshaving multi-well microtitre plate and/or multi-well sonicator.

In a still further embodiment of the invention, is provided an apparatusfor use in the methods of the invention or in the assays of theinvention. The apparatus of the invention comprises apparatus that havea microtitre plate and/or multi-well sonicator.

In a preferred embodiment, the prion disease is bovine spongiformencephalopathy (USE).

In a preferred embodiment, the prion disease is sporadic, variant,familial or iatrogenic Creutzfeld-Jacob Disease (CJD).

The present invention has been described with reference to the specificembodiments, but the content of the description comprises allmodifications and substitutions, which can be brought by a personskilled in the art without extending beyond the meaning and purpose ofthe claims.

The invention will now be described by means of the following Examples,which should not be construed as in any way limiting the presentinvention. The Examples will refer to the Figures specified here below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows in vitro prion replication on Hamster brain homogenate byPMCA assay in presence and absence of a cholesterol-depleting agent(Example 1§b). Samples contain 5% normal hamster brain homogenateincubated for 30 min at 4° C. with 0, 5, 10 or 20 mM (finalconcentration) of methyl-β-cyclodextrin (MβCD). Aliquots of scrapiebrain homogenate are added to reach a 3200- (top panel) and 12800-fold(bottom panel) dilution. Half of the samples are frozen immediately as acontrol without amplification (PMCA “−”) and the other half aresubjected to 10 cycles of PMCA (PMCA “+”). Prion replication isevaluated by Western Blot after treatment of the samples with PK (100μg/ml for 60 min). The first lanes in each blot corresponds to thenormal brain homogenate not treated with PK.

FIG. 2 shows the effect of Apolipoproteins B, E and J in vitro prionreplication on Hamster brain homogenate by PMCA assay Example 1§c).Samples containing 5% normal hamster brain homogenate are incubated withdifferent quantities of human Apolipoprotein B (2A), humanApolipoprotein E (2B) or murine Apolipoprotein J (2C) for 30 min at 4°C. Aliquots of scrapie brain homogenate are added to reach a 3200- (leftpanel) or 12800-fold (right panel) dilution. Half of the samples arefrozen immediately as a control without amplification (PMCA “−”) and theother half are subjected to 10 cycles of PMCA (PMCA “+”). Prionreplication is evaluated by western blot after treatment of the sampleswith PK (101 g/ml for 60 min). The first lanes in each blot correspondsto the normal brain homogenate not treated with PK.

FIG. 3 reports differential sensitivity of N2a sub-clones to infectionby Scrapie revealed by exposure to anti-PrP^(Sc) 6H4 mabs (Example n°2§b). Proteinase K (PK) exposure shows were the PrP^(Sc) isoform(Proteinase K resistant) is present. The two sub-clones highlighted #23and #60 are chosen respectively as representatives of prion infectionresistant and sensitive cells. ‘N2a’ shows uninfected N2a cellsprocessed in parallel. Controls for blotting and PK digestion show 1 μlnormal or scrapie brain extract diluted in 80 μl lysis buffer andprocessed in parallel.

FIG. 4 shows the characterization of PrP in lipid rafts from sub-clonesprion infection resistant (#23) and sensitive (#60) N2a (Example 2 §c).FIG. 4A shows PrP quantification by Western blotting in lipid raftswhich are extracted from prion infection resistant (#23) and sensitive(#60) cells. The distribution of PrP in the total extract (25 μg loaded)(1), the sucrose sample layer after centrifugation (25 μg loaded) (2)and the bouyant lipid raft fraction (4 μg loaded) (3) are presented.FIG. 4B shows PrP content and glycosylation pattern of the twosub-clones #23 and #60 by Western blotting with anti-PrP. Threeindependent preparations of lipid rafts prion infection resistant (#23)and sensitive (#60) cells were analysed. Equal amounts (4 μg) of raftsproteins were analysed in each case. FIG. 4C shows the same membraneafter stripping and re-probing with anti-actin which confirms thesimilarity in protein loading.

FIG. 5 presents the in vitro conversion activity of lipid rafts fromsub-clones prion infection resistant (#23) and sensitive (#60) N2a usingPMCA (Example 2 §d). Upper panel: Lipid rafts are isolated from prioninfection resistant (#23) and sensitive (#60) cells. Preparations aremixed in a ratio 100:1 with 10% RML brain homogenate and aliquots arefrozen immediately, incubated for 15 h at 37° C. or subjected to 15cycles of PMCA. Lanes 1: initial mixture without PK digestion; lanes 2:initial mixture digested 10 ug/ml PK 1 hr 37 C; lanes 3: mixtureincubated 37° C. PK digested as in lane 2; lanes 4: 15 cycles of PMCAfollowed by PK digestion as in lane 2. Lane 5 shows the migration andcross-reactivity with anti-PrP of PK alone. Lower panel: Followingwestern blotting the membrane is stained with Coomasie blue to confirmthat digestion with PK was complete.

FIG. 6 shows the inhibitory effect on Prion replication in prioninfection sensitive cells induced by Anti-hApoB polyclonal antibody(Example 2 §e).

Chronically infected #60 sensitive cells were cultured in 24 wellculture dishes in the presence of increasing amounts (0-2 mg/ml) of agoat polyclonal antibody against human ApoB (Chemicon) or against acorresponding series of naïve goat IgG. The level of PrP replication wasdetermined by quantitative dot blotting and expressed aschemiluminescent intensity/mg protein. In the graph, for each antibodyconcentration the chemiluminescent intensity is expressed as apercentage of the value obtained without the antibody. Higherconcentrations of anti-hApoB antibody have an inhibitory effect on PrPreplication.

FIG. 7 shows 2D separations of lipid raft proteins from N2a cells(Example 3). Lipid rafts are isolated from prion infection sensitivecells (#60) and 2 aliquots of 25 μg are precipitated with acetone andprocessed for 2D analysis min the 1st dimension spanning pH ranges 3-10(7A) or 6-11 (7B). After SDS-PAGE separation in the second dimension,gels are stained using the silver express kit (Invitrogen). Arrowindicates the same protein on both gels (7A and 7B). Proteins within therectangle shown in B are compared between lipid raft from the prioninfection sensitive sub-clone #60, (C) and resistant subclone #23, (D).Arrows indicate proteins which am more abundant in resistant cells.

Abbreviations:

Apo B (Apolipoprotein B; Apo E (apolipoprotein E); Apo J (Apolipoprotein3); BCA (Bicinchoninic Acid); CHAPS(3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate); CNS(central nervous system); BSE (bovine spongiform encephalopathy); CJD(Creutzfeldt-Jakob Disease); DIM(1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate); DIM(Detergent-Insoluble Membrane); DMEM (Dulbecco's Modified Eagle Medium);DRM (Detergent-Resistant Membrane); DTT (1,4-Dithio-D,L-threitol); IPG(Immobilized PH Gradient); IEF (Isoelectric Focusing); FCS (Fetal CalfSerum); FFI (Fatal Familial Insomnia); GSS(Gerstmann-Strassler-Scheinker Disease); hr (hour); HRP (HorseradishPeroxidase); kDa (KiloDalton); LDL (Low Density Lipoprotein); μg(microgram); μl (microliter); min (minute); MβCD(methyl-β-cyclodextrin); mM (millimolar); MS (mass spectrometry); PBS(Phosphate Buffered Sulfate); PK (proteinase K); PMCA (ProteinMisfolding Cyclic Amplification); PMSP (Phenylmethanesulfonyl Fluoride);PrP (prion protein); PrP^(C) (normal, non-pathogenic conformer of PrP);PrP^(Sc) (pathogenic or “scrapie” isoform of PrP which is also themarker for prion diseases); PVDF (polyvinylidene difluoride); RPM(Rotation per minute); RML (Rocky Mountain Laboratory); RT-PCR (reversetranscriptase polymerase chain reaction); SDS (Sodium Dodecyl Sulfate);V (Volt); Vol. (volume).

EXAMPLES

The invention will be illustrated by means of the following exampleswhich are not to be construed as limiting the scope of the invention.

The following examples illustrate preferred compounds and methods fordetermining their biological activities.

PrP scrapie used as infection innoculum is RML (Rocky MountainLaboratory) strain.

Anti-PrP 6114 monoclonal antibodies were purchased from Prionics.

Proteinase K was obtained from Boerhinger Ingelheim andMethyl.β.cyclodextrin from Sigma.

Purified and delipidated human Apolipoprotein B (Apo B) andApolipoprotein B (Apo E) were obtained from Chemicon.

Anti-apo B and anti-apo E are goat polyclonal antibodies against humanApo B and human Apo E, respectively obtained from Chemicon and dialysedagainst PBS to eliminate sodium azide.

Total goat IgG was purchased from Pierce and dialyzed against PBS.

Mouse neuroblasma N2a cell line was obtained from ATCC.

Murine Apo J (Apo J) was obtained in-house as described inPCT/EP2004/05037.

DiI labeled LDL was obtained from Molecular Probes (L-3482).

Example 1 In vitro Prion Replication in Brain Homogenate through PMCAAssay

The influence of cholesterol and some of the apolipoproproteins on prionreplication in vitro is analysed through a Protein Misfolding CyclicAmplification assay (PMCA) (Saborio et al., 2001) where hamster brainhomogenate is used as a source of PrP^(C) and conversion factors asfollows.

a) Brain preparation:

Brains from healthy Syrian golden hamsters healthy or infected with theadapted scrapie strain 263 K are obtained after decapitation andimmediately frozen in dry ice and kept at −0′ until used. Brains arehomogenized in PBS containing protease inhibitors (Complete™ cocktailfrom Boehringer Mannheim) at a 1× final concentration. Detergents (0.5%Triton X-100, 0.05% SDS, final concentrations) are added and samplesclarified with low speed centrifugation (10000 g) for 1 min, using anEppendorf centrifuge (model 5415).

Dilutions (3200-fold and 12800-fold) of the scrapie brain homogenate areadded directly to the healthy brain homogenate to trigger prionreplication. 60 μl of these mixtures are frozen immediately and another60 μl are incubated at 37° C. with agitation. Each hour a cycle ofsonication (5 pulses of 1 sec each) is done using a microsonicator(Bandelin Electronic, model Sonopuls) with the probe immersed in thesample and the power setting fixed at 40%. These cycles are repeated 10times.

b) PMCA signal in presence and absence of a cholesterol-depleting agent:

Under these conditions a dramatic increase in the amount of PrP^(Sc)signal is observed after 10 cycles of PMCA (FIG. 1, lanes 2 and 3). Whennormal brain homogenate is treated during 30 min with 10 and 20 mM (butnot 5 mM) methyl-β-cyclodextrin (13CD) a complete inhibition of prionreplication is observed (FIG. 1, lanes 6-9) as obtained in mouse modelsin cell cultures and in vitro, indicating that cholesterol depletion hasa detrimental effect on prion replication (Taraboulos et al., 1995).

c) PMCA signal In presence of apolipoproteins

Purified delipidated human ApoB (FIG. 2A) and human Apo E (FIG. 2B) arerespectively added to the PMCA preparation without cyclodextrin atdifferent concentrations (8 and 16 μg for hApo B) and (1 and 10 μg forhApo E). Samples are incubated for 30 min at 4° C. and thereafter halfof each sample is frozen and the other half subjected to PMCA cycles.

An increase in prion replication in vitro is observed at both 3200-foldand 12800-fold dilutions of scrapie brain homogenate for bothApolipoprotein B and Apolipoprotein E.

In contrast, addition of Apolipoprotein J (at concentrations of 1, 2 and4 μg), an Apolipoprotein component of HDL, has no effect on PMCA signal(FIG. 2C).

These data show the effect of Apolipoprotein B and E implicated in theprion conversion.

Example 2 In vitro Prion Replication in Lipid Rafts from Prion InfectionSensitive Cells by PMCA

The mouse neuroblastoma cell line N2a is used for their capability to beinfected with PrP^(Sc). Baron et al., 2002 and Enari et al., 2001 haveshown that prion infection sensitive and prion infection resistant N2asub-lines exist. Lipid rafts from the prion infection sensitive line areisolated and used as a substrate for PMCA assay. The effect of anapolipoprotein B antagonist on prion conversion is studied through theability of apolipoprotein B antagonist to inhibit the prion replicationability of prion infection sensitive N2a cell lines.

a) N2a cell preparation:

Sub-clones of the parental mouse neuroblastoma N2a cell line are derivedfrom single cells by limit dilution. A growing culture (Dulbecco'sModified Eagles Medium (DMEM Gibco # 41966-029), containing 10% fetalcalf serum (FCS) and supplemented with 2 mM, L-glutamate and standardantibiotics (penicillin and streptomycin)) is diluted to a density of 5cells/ml and 100 μl is transferred to individual wells of a 96 wellplate and cultured for 1 week.

The individual cultures are examined microscopically to determine thosewells which contained a single focus of growing cells. The single cellderived cultures are then transferred to 24 well plates and seriallypassaged every 3-4 days at 1:15 dilution to maintain stocks. A total of63 cultures are isolated and all tested for sensitivity to infection bythe RML strain of PrP^(Sc). To do this, 4 μl of a 10% late stageinfected brain extract is added per well of newly passaged cells, andthe cultures are left for a further 4 days to reach 100% confluence.Cells were serially passaged thereafter in the absence of PrP^(Sc).Tests showed that all trace of the initial innoculum disappeared bypassage 4.

At this and later passages individual cultures are tested for thepresence of PrP^(Sc).

b) Prion infection resistant cell isolation by Cell culture dotblotting:

The presence of PrP^(Sc) in the 63 individual cell cultures is tested bycell culture dot blotting procedure in which lysis and proteinase K (PK)digestion are carried out directly in the culture dish. PK resistantPrP^(Sc) is detected by dot blotting to PVDF membranes and exposure toanti-PrP antibody as follows:

Infected cells are grown for 3-4 days in 24-well plates and washed oncewith PBS. 40 μl DNase1 (1000 U/ml in H2O) is added to each well at roomtemperature for 5 min, followed by 40 μl proteinase K solution (20 μg/mlin 100 mM Tris/HCl pH 7.4, 300 mM NaCl, 1% Triton-X100, 1% sodiumdeoxycholate). Plates are incubated at 37° C. for 1 hr with gentleagitation. Proteinase K digestion is stopped by addition of 2 μl of asolution containing 80 μg/ml PMSF, 10 mM Tris HCl pH 8.0 and 1 mg/mlbromophenol blue. 20 μl aliquots are spotted onto PVDF membranesequilibrated with a degasses solution containing 192 mM Glycine, 25 mMTris, 20% methanol. Membranes are then transferred to 3M guanidineThiocyanate, 10 mM Tris HCL pH 8.0 for 10 min to denature proteins,rinsed in water and processed as for Western blotting using anti-PrP 6H4(Prionics). Non-specific binding is blocked by incubation with 5% milkdissolved in PBS for 1 hr. The membrane is then exposed to specificprimary antibody anti-PrP 6H4, followed by HRP-conjugated secondaryantibody each diluted as appropriate in PBS, 0.3% Tween 20. Westernblots are developed by ECL™ (Amersham) as directed according to theprovider instructions.

The chemiluminescence signal from membranes is then analyzed directlyusing the Kodak Imagestation 440CF. The luminescence signal in eachcondition was normalized for possible differences in cell growth Totalprotein content of a parallel lysate untreated with proteinase K isdetermined using the BCA assay (Biorad) and results are expressed asintensity/μg protein.

Of the 63 sub-clones analysed, 9 were found to be capable of replicatingPrP^(Sc), albeit with differing efficiencies (FIG. 3). The remaining 54sub-clones were resistant to infection. The most highly prion infectionsensitive cell lines were selected for further study together withseveral prion infection resistant sub-clones with similar morphologiesand doubling times. We have have selected two of these cell lines: #23,a prion infection resistant clone, and #60 a prion infection sensitiveclone.

These two cell lines have been maintained in culture for over 1 year andhave been infected with RML in many different occasions throughout thisperiod: on every occasion sub-clone #60 was highly infectable whereassub-clone #23 was totally resistant. Prion infection sensitivesub-clones could be maintained as a chronically infected cell culture byserial passaging at 1:15 or 1:20 dilution every 3 or 4 daysrespectively.

No evident morphological differences by microscopy were observed betweenthe resistant or sensitive cells or between non-infected and infectedcells.

To validate the clinical relevance of this cellular model of PrPrelication, exacts of chronically infected N2a cells, or buffer alone,were injected into the hippocampus of normal mice by stereotacticinjection. Injection of N2a extracts resulted in onset of clinicalsymptoms of scrapie after 140 days and premature death whereas mockinjection had no effect on mouse physiology or life span. This indicatesthat the cell based model for prion replication using prion infectionsensitive N2a cells generates infectious PrP scrapie, confirming thatthe conversion of PrP in cells is a good model for the process whichoccurs in vivo.

c) Lipid rafts isolation:

Procedures for isolating lipid rafts based on resistance tosolubilization in cold Triton X-100 followed by flotation on sucrosegradients have been described by numerous laboratories (Simons et al.,2000; Hooper et al., 1999). Lipid rafts from the two cell lines selectedabove are carried out as follows:

Subconfluent cultures of N2a cells are washed in PBS and collected bycentrifugation 1000×g for 5 min. Typically 3×15 cm dishes are pooledequivalent to approximately 8×10⁷ cells. The cell pellet is re-suspendedin 1 ml ice cold raft buffer (1% Triton in PBS, containing 10 μM coppersulphate and a cocktail of complete protease inhibitors (Boehringer)).Cells are disrupted by seven passages through a 22G needle followed byincubation for 30 min at 4° C. with gentle agitation 2 volumes 60%sucrose in PBS is added and the lysate is transferred to a SW41centrifuge tube. The lysate is carefully overlaid with 7 ml 35% sucroseand 1 ml 15% sucrose both in PBS and centrifuged 20 hr at 35,000 RPM.The lipid rafts are recovered in the top 1 ml of the gradient Membranesare concentrated by addition of 10 volumes cold PBS and centrifugationat 100,000 g for 2 hr. Alternatively for 2D gel electrophoresis,proteins from the lipid raft fraction are recovered by precipitation inthe presence of 5 vol acetone for 2 hr at −80° C. Acetone precipitatesare collected by centrifugation 14000 g 20 min and washed twice in 70%ethanol.

In both sensitive and resistant cells around 1-2% of protein in thetotal lysate is recovered in the bouyant raft fraction. As shown byWestern blotting (FIG. 4A) while PrP is barely detectable in the totalcell extract, it is highly enriched in rafts leaving the sample layertotally depleted of PrP following centrifugation.

Prion infection sensitive clone #60 and the prion infection resistantclone #23 are compared by western blotting with anti-PrP (FIG. 4B).Three different independent pairs of raft preparations each containing 5μg total raft proteins are re-probed with anti-actin antibody whichconfirms the uniformity of PrP protein loading (FIG. 4C).

The results indicate that the level of PrP in the lipid raftpreparations from the two cell types is indistinguishable. Moreover thedistribution between non-glycosylated mono- and di-glycosylated isoformsas well as the segregation to the detergent resistant membrane fractionshown in FIG. 4A is identical suggesting that none of these factors arelikely to be responsible for the differing phenotypes.

PrP cDNA was amplified by RT-PCR from both cell lines as follows:

Total RNA of N2a cells is prepared using Trizol (Gibco) and the mousePrP cDNA is reversed transcribed with Omniscript (Qiagen) using theprotocol supplied by the manufacturer. The specific primer for cDNAsynthesis is 5′ TCAATTGAAAGAGCTACAGGTG 3′. The prion cDNA is amplifiedusing standard PCR conditions in the presence of primers 5′ACCAGTCCAATTTAGGAGAGCC 3′ (top strand) and 5′ AGACCACGAGAATGCGAAGG 3′(bottom strand). The PCR product was completely sequenced in theautomated ABI3700 using the reagents and the protocol supplied by themanufacturer.

These data revealed that PrP mRNA is wild type in both cases and thatboth carry a Methionine at position 129, which in humans is the site fora frequent polymorphism.

Therefore, the expression levels, glycosylation patterns, intracellularlocalisation and primary sequences of PrP^(C) in both cell types isindistinguishable and thus that other cellular factors are responsiblefor the differential response to PrP^(Sc).

d) In vitro cyclic amplification of protein misfolding (PMCA) in lipidrafts from prion infection sensitive cells:

Lipid rafts obtained at §c are isolated from prion infection sensitivesub-clones, #60 sub-clones, collected by centrifugation as describedabove and re-suspended in PMCA conversion buffer at a concentration of2-2.5 mg/ml (PBS containing final concentration of 300 mM NaCl, 0.5%Triton X100, 0.05% SDS).

A 10% extract of RML-infected mouse brain homogenate is added directlyto the rafts preparation at a dilution of 1:100 based on protein contentand aliquots of the mixture are either frozen immediately, incubated for15 hr at 37° C. or subjected to 15 cycles of PMCA (5×0.1 second pulsesof sonication followed by incubation at 37° C. for 1 hr).

Aliquots of 20 μl sample are then treated with 10 μg/ml Proteinase K for1 hr at 37° C. Lipids are removed by precipitating PK-resistant proteinswith 5 vol acetone for 2 hr at −80° C. Acetone precipitates arecollected by centrifugation 14000 g 20 min, washed twice in 70% ethanolanalysed by Western blotting with 6H4 anti-PrP (FIG. 5).

Compared to the mixture without PK treatment (lanes 1 and 5) alldigested samples show a shift in molecular weight characteristic of theN-terminally truncated PK resistant form PrP₂₇₋₃₀. It should be notedthat the 6H4 antibody also has low level cross reactivity with PK whichmigrates at 30 kDa, close to the di-glycosylated form of PK-digestedPrP. Analysis of the data with this in mind shows that the initial levelof PK-resistant PrP derived from the diluted brain extract, which ispresent in the non-amplified mixtures, is barely detectable under theseconditions (lanes 2).

A slight increase in signal is seen when the prion infection sensitive(#60) DRM is incubated at 37° C. for 15 hr (lane 3 from #60), howeverthe most dramatic increase in PK-resistant PrP is seen when this sampleis subjected to 15 cycles of PMCA (lane 4 from #60). This indicates thatall factors required for conversion of PrP^(C) to PrP^(Sc) are residentin the lipid rafts from the prion infection sensitive N2a cells.Interestingly, in the parallel analysis in which the DRM from the prioninfection resistant cell line #23 was used, no amplification in vitrowas observed (lane 4 from #23) indicating that the capacity of the lipidrafts to convert the prion protein in vitro reflects the activityobserved in the intact cells.

e) Effect of antibody raised against apolipoprotein B on prionreplication by prion infection sensitive N2a cells:

Chronically infected sensitive cells were cultured in 24-well dishes inthe presence of a goat polyclonal antibody raised against human Apo B(Chemicon) at increasing concentrations from 0 to 2 mg/ml in DMEM Gibco# 41966-029, containing 1× B27 supplements (Gibco #17504-044) andstandard antibiotics (penicillin and streptomycin)

A parallel series of cultures was incubated in the presence of the sameconcentration range of total IgG from a naïve goat. The results showthat concentrations of anti-hApoB antibody above 0.5 mg/ml result inprogressive inhibition of PrP replication as revealed by quantitativedot blotting (FIG. 6).

These data show the role of Apolipoprotein B in the prion conversion.

Example 3 Proteomics Analysis of Lipid Rafts of Prion InfectionResistant and Sensitive Cells

Since the two cell preparations are indistinguishable in terms of theirPrP content a more complete protein comparison using 2D gelelectrophoresis was performed to show differences in other proteins thatmight underline the difference in conversion activity between the twosub-clones.

2D gel preparations are prepared as follows:

Acetone precipitated proteins (see §c) are re-suspended in 20 μl 1% SDS,0.23% DTT and heated to 95° C. for 5 min After the preparation is cooledto room temperature, 25 μl of a solution (9M urea, 4% CHAPS, 65 mM DTT,35 mM Tris base) is added.

Fifteen minutes later, 85 μl of a solution containing 7M urea, 2Mthiourea, 4% CHAPS, 100 mM DTT is further added to the mixture. After afurther 15 min, non-solubilized material is removed by centrifugation at14000 RPM during 5 min and the supernatant is applied directly to a 7 cmIPG strip and left to re-hydrate overnight. For IEF the voltage isprogressively increased from 300V to 3.5 kV and electrophoresed for atotal of 20 kVh. Proteins are resolved in the second dimension usingsingle well 4-12% gradient gels (Novex) and stained using the silverexpress kit (Invitogen) according to the instructions supplied.

Analysis by 2D gels reveals the fraction of protein that is recovered inthe lipid rafts (approximately 1-2% protein in the N2a cell lysate) as areproducible subset of total cell proteins in which several hundredspecies can be visualized following silver staining (FIGS. 7A and B).

The 2D patterns are compared between preparations isolated from theprion infection sensitive and resistant cells. The analysis is focusedon several proteins identified in the basic range of the gel which aremore abundant in DRMs from prion infection resistant cells (arrows inFIGS. 7C and D).

Following preparative scale electrophoresis, the two proteins indicatedby arrows are excised and processed for MS sequencing. From bothproteins an identical tryptic peptide is found with a monoisotopic massof 1234.6. The N-terminal sequence of this tryptic peptide is:ENFAGEATLQR (SEQ ID NO: 3). AR amino acids in the peptide are identifiedin the MS/MS spectrum of doubly charged precursor ion at m/z 618.30 Andthrough its Mascot analysis.

Database searching identified this protein unambiguously asApolipoprotein B (Apo B). Since the molecular weight of fall length ApoB is in excess of 500 kDa while these two spots migrate with estimatedmolecular weights of 40kDa and 30 kDa, we presume that the latter arefragments generated either in the cell or during sample preparation. Thesequence corresponds to amino acids 3548-3558 of the human Apo Bprotein, which is present only in ApoB-100 and not in the truncatedApoB-48 form.

These data suggest that fragments of a molecular weight of or about 30to 40 kDa comprising the sequence of SEQ ID NO: 3 may have a role in theprion conversion pathway.

Example 4 Binding and Internalisation of Fluorescent LDL Receptor byResistant and Sensitive Cells

N2a subclones #23 (prion infection sensitive) and #60 (prion infectionresistant) were cultured in 24 well plates in standard DMEM mediumcontaining 10% FCS for 2 days then transferred to the same medium (300μl) containing 1% FCS for 1 hr. To visualize cell surface binding,plates were placed on ice to inhibit endocytosis and 3 μl fluorescentDiI-LDL (Molecular Probes) was added for 30 min.

LDL-binding was visualized by fluorescence microscopy. To study LDLuptake by each of the sub-clones, cells were incubated at 37 C with 3 μlDiI-LDL for 2 h prior to microscopic examination.

Control cultures were incubated in parallel with DiI-coupled acetylatedLDL which does not bind the LDL receptor or with Hoechst to visualizecell nuclei.

The binding or uptake of fluorescent DiI-LDL is similar for prioninfection resistant and prion infection sensitive cells, suggesting thatthe level of the LDL receptor between these two cell types is similar.

REFERENCES

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1-14. (canceled)
 15. A method for the diagnosis or detection of a priondisease within a subject suspected of suffering from such a disease, themethod comprising: (i) contacting a sample from said subject with apeptide or a protein selected from the group consisting ofApolipoprotein B; a fragment of Apolipoprotein B; Apolipoprotein E; anda fragment of Apolipoprotein E; (ii) contacting the preparation obtainedin step (i) with PrP^(C) or PrP^(C) containing mixtures; and (iii)determining the presence and/or an amount of PrP^(Sc) in said sample.16. A method of determining the presence of a marker that predisposes asubject to a prion disease, the method comprising: (i) measuring a levelof a protein of Apolipoprotein B or a fragment thereof in said sample;and (ii) correlating said level of protein obtained in said measuringstep with the occurrence of the prion disease.
 17. The method of claim15, wherein the prion disease is bovine spongiform encephalopathy (BSE).18. The method of claim 15, wherein the prion disease is aCreutzfeld-Jacob disease.
 19. A method for the detection of PrP^(Sc)within a sample, the method comprising: (i) contacting said sample witha peptide or a protein selected from the group consisting ofApolipoprotein B; a fragment of Apolipoprotein B; Apolipoprotein E; anda fragment of Apolipoprotein E; (ii) contacting the sample obtained in(i) with PrP^(C) or PrP^(C) containing mixtures; and (iii) determiningthe presence and/or an amount of PrP^(Sc) in said sample.
 20. A methodfor identifying, in a sample, a compound which modulates the transitionof PrP^(C) into PrP^(Sc) the method comprising: (i) contacting saidsample with a peptide or a protein selected from the group consisting ofApolipoprotein B; a fragment of Apolipoprotein B; Apolipoprotein E; anda fragment of Apolipoprotein E (a) in the presence of said modulatorycompound and (b) in the absence of said compound; (ii) contacting thepreparation obtained in step (i) a and (i) b with PrP^(C) or PrP^(C)containing mixtures; and (iii) determining the amount of PrP^(Sc) (a) inthe presence of said modulatory compound and (b) in the absence of saidmodulatory compound.
 21. The method of claim 15, wherein the peptide orthe protein contains the sequence of SEQ ID NO:
 3. 22. The method ofclaim 15, wherein the peptide or the protein has a molecular weight from30 and 40 kDa and has a sequence obtained from fragments ofApolipoprotein B between positions 3201-3558, 3548-3905, 3201-3905,3291-3558, 3548-3815, and 3291-3815. 23-26. (canceled)
 27. A diagnostickit for the detection of PrP^(Sc) within a sample, the kit comprising aprobe for receiving a sample; and a peptide or a protein selected fromApolipoprotein B and a fragment thereof.
 28. (canceled)
 29. The methodof claim 15, wherein the protein is Apolipoprotein B or a fragmentthereof.
 30. The method of claim 15, wherein the peptide or the proteinforms a complex with a LDL receptor.
 31. The method of claim 15, whereinthe peptide or the protein contains the sequence of SEQ ID NO:
 3. 32.The method of claim 15, wherein the peptide or the protein has amolecular weight from 30 and 40 kDa and has a sequence obtained fromfragments of Apolipoprotein B between positions 3201-3558, 3548-3905,3201-3905, 3291-3558, 3548-3815, and 3291-3815.
 33. The method of claim16, wherein the prion disease is selected from the group consisting ofbovine spongiform encephalopathy (BSE) and a Creutzfeld-Jacob Disease(CJD).
 34. The method of claim 19, wherein the protein is ApolipoproteinB or a fragment thereof.
 35. The method of claim 19, wherein the peptideor the protein forms a complex with a LDL receptor.
 36. The method ofclaim 19, wherein the peptide or the protein contains the sequence ofSEQ ID NO:
 3. 37. The method of claim 19, wherein the peptide or theprotein has a molecular weight from 30 and 40 kDa and has a sequenceobtained from fragments of Apolipoprotein B between positions 3201-3558,3548-3905, 3201-3905, 3291-3558, 3548-3815, and 3291-3815.
 38. Themethod of claim 19, wherein the prion disease is selected from the groupconsisting of bovine spongiform encephalopathy (BSE) and aCreutzfeld-Jacob Disease (CJD).
 39. The method of claim 20, wherein theprotein is Apolipoprotein B or a fragment thereof.
 40. The method ofclaim 20, wherein the peptide or the protein forms a complex with a LDLreceptor.
 41. The method of claim 20, wherein the peptide or the proteincontains the sequence of SEQ ID NO:
 3. 42. The method of claim 20,wherein the peptide or the protein has a molecular weight from 30 and 40kDa and has a sequence obtained from fragments of Apolipoprotein Bbetween positions 3201-3558, 3548-3905, 3201-3905, 3291-3558, 3548-3815,and 3291-3815.
 43. The method of claim 20, wherein the prion disease isselected from the group consisting of bovine spongiform encephalopathy(BSE) and a Creutzfeld-Jacob Disease (CJD).
 44. The method of claim 20,wherein determining the amount of PrP^(Sc) in the sample comprisesperforming a Protein Misfolding Cyclic Amplification (PMCA) assay. 45.The method of claim 44, wherein the sample is a normal brain homogenateas a source of normal PrP^(C) and substrate.
 46. The method of claim 44,wherein the sample is lipid rafts from an infection-sensitiveneuroblasma cell line N2a as a source of normal PrP^(C) and substrate.47. The method of claim 20, wherein the protein is Apolipoprotein B,determining the amount of PrP^(Sc) in the sample comprises performing aProtein Misfolding Cyclic Amplification (PMCA) assay, and the sample islipid rafts from infection sensitive neuroblasma cell line N2a as asource of normal PrP^(C) and substrate.
 48. The method of claim 20,wherein said modulatory compound is an antagonist of Apolipoprotein B ora fragment thereof.
 49. The method of claim 20, wherein said modulatorycompound is an antibody raised against Apolipoprotein B or a fragmentthereof.
 50. The method of claim 20, wherein said modulatory compound isa LDL-receptor antagonist.
 51. A method for treatment of a priondisease, comprising: administering a modulator of Apolipoprotein B or afragment thereof to a subject in an amount sufficient to treat the priondisease.
 52. The method of claim 51, wherein the modulator is anantagonist of Apolipoprotein B or a fragment thereof.
 53. The method ofclaim 51, wherein the modulator is an antibody raised againstApolipoprotein B or a fragment thereof.
 54. The method of claim 52,wherein the antagonist is a peptide or a protein that contains thesequence of SEQ ID NO:
 3. 55. The method of claim 52, wherein theantagonist is a peptide or a protein that has a molecular weight from 30and 40 kDa and has a sequence obtained from fragments of ApolipoproteinB between positions 3201-3558, 3548-3905, 3201-3905, 3291-3558,3548-3815, and 3291-3815.
 56. The method of claim 51, wherein themodulator is an antagonist of a LDL-receptor.
 57. The method of claim51, wherein the prion disease is selected from the group consisting ofbovine spongiform encephalopathy (BSE) and a Creutzfeld-Jacob Disease(CJD).